Indazole and its Derivatives in Cardiovascular Diseases: Overview, Current Scenario, and Future Perspectives.

Indazole derivatives are currently being explored as drug molecules in both preclinical and clinical studies ( Tables 1 and 2 ). There is a huge scope to explore indazole derivatives for further development in cardiovascular as well as other diseases. As previously reported, ARRY-371797/ARRY-797 is a highly potent and selective p38ą (MAPK14) kinase inhibitor. According to previous studies, the p38 mitogenactivated protein kinase (MAPK) plays a key role in ischemic heart disease, right ventricular hypertrophy, and many other cardiac diseases. According to Kojonazarov et al., phosphorylated p38 MAPK is significantly enhanced in hypertrophied/failed right ventricles in both mice and humans. Inhibition of p38 MAPK may reduce collagen production and stress fibre formation in right ventricular fibroblasts. ARRY-371797/ARRY-797 also prevents the differentiation of right ventricular fibroblasts into myofibroblasts by inhibiting myocardin-related transcription factor A (MRTF-A), translocation of MRTF-A from the cytosol to the nucleus. p38 MAPK is considered as a potential drug target for the reduction of endothelial dysfunction, increased fibroblast activity, inflammation, and right ventricular failure [ 70 ]. Based on the previous studies, inhibition of p38 with indazole derivative ARRY-371797/ARRY-797 may show beneficial effects on cardiac diseases. The beneficial effect of In-Cl could be attributed to its effect in reducing PDGF expression. Heldin et al. reviewed that both PDGF-AA and PDGF-BB in mouse hearts cause cardiac fibrosis with the increased collagen deposition by interstitial cardiac fibroblasts [ 71 ]. Park et al. reported that PDGF-BB treatment increased the cell proliferation in the vascular smooth muscle cell, and this was normalised with In-Cl treatment. Therefore, targeting PDGF with indazole derivatives could be a future strategy to reduce cardiac remodelling and heart failure. Further basic and clinical research should be undertaken to investigate the beneficial effect of indazole derivatives in cardiovascular diseases. Though there are several studies that indicate the beneficial effects of indazole derivatives, there is only one toxicity study with one indazole derivative, i.e., 7-nitroindazole. The study reported that the administration of 7-nitroindazole to Wistar rats from the prenatal period to adulthood resulted in a reduction in the weight of the heart and kidneys, along with reduction of wall thickness/inner diameter ratio in the thoracic aorta and carotid artery [ 56 ]. The study indicates the requirement to conduct more toxicity studies to evaluate the adverse effect of other indazole derivatives.

Indazole derivatives are being used as drug molecules in various diseases including cancer, and cataracts ( Table 3 ). Many indazole derivatives have shown potential beneficial effects against cardiovascular and metabolic diseases such as arrhythmia, ischemia-reperfusion injury, thrombosis, hypertension, hyperlipidemia, and obesity. One of the most promising indazole derivatives, DY-9760e, has shown cardioprotection against ischemic/reperfusion injury [ 22 ]. Another promising indazole derivative, YC-1, has been developed for its therapeutic use in circulatory disorders, platelet aggregation, and vascular contraction [ 35 ]. YC-1 has been identified as an activator of the physiological nitric oxide receptor, soluble guanylyl cyclase, a signalling molecule in the cardiovascular system [ 72 ]. Another indazole derivative, indazole-Cl, suppressed the inflammation in atherosclerosis [ 44 ]. 7-nitroindazole has an anti-hypertrophic effect on the heart and reduces wall thickness of the thoracic aorta and carotid arteries [ 56 ]. Few indazole derivatives are in clinical trials and have shown promising results against cardiovascular diseases. ARRY-371797, a p38α (MAPK14)-selective kinase inhibitor, is now being tested in humans for LMNA-related dilated cardiomyopathy ( NCT02351856 , NCT03439514 ) [ 36 , 37 ]. Indazole derivatives need to be explored further by researchers in academic institutes and pharma industries to develop novel drug molecules against newer targets in several cardiovascular diseases where adequate treatment is not available.

Indazoles, a class of heterocyclic compounds with a bicyclic ring structure composed of a pyrazole ring and a benzene ring, have a wide range of biological and pharmacological applications. Indazole was first defined by scientist Emil Fisher as a “pyrazole ring fused with the benzene ring.” Indazoles belong to the azoles family and contain carbon, hydrogen, and nitrogen atoms. There are generally two tautomeric variants of indazole, 1H-indazole, and 2H-indazole ( Fig. 1 ). The main tautomer is 1H-indazole, which is thermodynamically more stable than 2H-indazole [ 1 ]. Because of its unique chemical and biological features, indazole derivatives have been intensively researched. This nucleus, found in a number of synthetic compounds, has a wide range of pharmacological properties, e.g., metabolic (antihyperlipidemic, anti-obesity), cardiovascular (antiarrhythmic, antihypertensive), antimicrobial (antifungal, anti-bacterial, trichomonacidal), central nervous system (CNS) (antipsychotic, analgesic and antipyretic), anticancer as well as other activities ( e.g. against rheumatoid arthritis) [ 2 - 15 ].

Indazole derivatives ( Table 1 ) are important pharmacological agents and form the basic structure of several drug molecules. Indazole derivatives are building blocks of numerous therapeutic compounds as well, such as edaravone (MCI-186) for the treatment of amyotrophic lateral sclerosis (ALS) ( NCT00424463 , NCT00424463 ) [ 16 ]. Other examples of indazole derivatives are granisetron and benzydamine used as 5HT 3 receptor antagonists (as an anti-emetic) and an anti-inflammatory agent, respectively. The indazole derivatives can be functionalized with high selectivity at different positions. Changing the planarity of the indazole ring, modifying the structure with side chain, and changing their length at different positions can afford a huge number of indazole derivatives, providing novel molecules with varieties of therapeutic properties. We will discuss different indazole derivatives obtained from natural products and synthetic sources. Nigella sativa (family: Ranunculaceae) is commonly known as black seed [ 17 ]. The extracts from N. sativa have been extensively explored for their therapeutic potential and shown to possess a wide spectrum of activities, viz. antihypertensive, bronchodilator, diuretic, renal protective, and antioxidant properties. Seeds from Nigella glandulifera contain pyrazole alkaloids or indazole ring-bearing alkaloids which include nigellicine, nigellidine, and nigeglanine ( Fig. 2 ) [ 18 - 20 ]. The only natural source of indazole alkaloids is nigella species [ 21 ]. DY-9760e, 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl] ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate, is a novel calmodulin antagonist ( Fig. 3 ) [ 22 ]. DY-9760e has shown cardioprotection from ischemic/reperfusion injury. The activation of the CaMKII (Ca 2+ /calmodulin-dependent protein kinase II) is essential for myocardial contraction. Alteration of CaMKII activity in cardiomyocytes may result in myocardial disorder [ 23 , 24 ]. For example, excessive activation of CaMKII can be characterized by increased activity of Ca 2+ channel gating, leakage of Ca 2+ from sarcoplasmic reticulum, and dysregulation of Ca 2+ homeostasis, which may together cause arrhythmia and heart failure. Hashimoto et al. reported that increased levels of intracellular calcium due to ischemia activate the Ca 2+ dependent calpains, also known as cysteine proteases, leading to degradation of structural proteins such as fodrin, thereby resulting in contractile dysfunction in the rat heart [ 22 ]. The molecule DY-9760e inhibits the degradation of the cytoskeletal protein fodrin and protects the heart from further damage. In general, fodrin binds to actin, calmodulin, and microtubules and is attached to the plasma membrane. Fodrin plays an important role in organelle organisation, molecular transportation, and secretion [ 25 ]. Fodrin is known to be proteolyzed by both calpains and caspase-3 [ 26 ]. Caspase-3 activation has a crucial role in the apoptosis process in the cardiomyocytes. Caspase-3 activity was dramatically decreased by DY-9760e therapy and thereby exerts cytoprotective effects by inhibiting fodrin degradation and tyrosine nitration in cardiac proteins ( Fig. 4 ). DY-9760e has no effect on calpains, which are calmodulin (CaM)-independent enzymes [ 27 ]. CaM has been shown to stimulate calpain’s degradation of fodrin, but DY-9760e inhibits fodrin proteolysis by inhibiting CaM rather than calpains [ 28 ]. DY-9760e also protects dystrophin from degradation. Dystrophin is an essential protein for maintaining membrane structural integrity in cardiac and skeletal myocytes [ 29 ]. Calpain activation increases the breakdown of dystrophin and leads to myocardial injury [ 30 ]. This injury is restored by DY-9760e treatment [ 31 ]. DY-9760e has been shown to be neuroprotective and reduces infarct size in the permanent focal ischemia model of spontaneously hypertensive rats by decreasing proteolysis of fodrin [ 28 ]. DY-9836 is an active metabolite of DY-9760e. It has shown antioxidant properties by inhibiting the elevated levels of both superoxide and nitric oxide (NO) production in cultured cardiomyocytes [ 32 ]. The expression of hypertrophy-related genes such as atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) was elevated by endothelin-1-induced cardiomyocyte hypertrophy. These gene expressions were dramatically reduced by DY-9760e treatment [ 31 , 33 , 34 ]. Caveolin-3 is a caveolin component found in cardiac and skeletal muscle caveolae. Caveolin-3 is a scaffolding protein that interacts with and organises lipid and protein constituents in caveolae, including endothelial nitric-oxide synthase (eNOS). It is found in the sarcolemma and plays a role in the formation of caveolae membranes. Caveolin-3 binds to eNOS in caveolae and prevents it from generating NO. Feng Han et al. reported that significant transverse aortic constriction induced caveolin-3 breakdown in the left ventricle compared with control animals. This effect was significantly inhibited by DY-9760e treatment [ 31 , 33 , 34 ]. Phenylephrine-induced hypertrophy is associated with a caveolin-3 breakdown in cultured cardiomyocytes [ 34 ]. Cardiac-specific expression of caveolin-3 attenuates cardiac hypertrophy. DY-9760e inhibits the degradation of caveolin-3 and thereby shows its cardioprotection. Overall, DY-9760e, a novel calmodulin antagonist, effectively blocks activation of both calpain and caspase 3, and inhibits the degradation of fodrin, dystrophin, and caveolin-3 to provide its cardioprotective effect. YC-1, 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole, ( Fig. 5 ) is a molecule developed for the treatment of circulatory disorders. YC-1 inhibits platelet aggregation and vascular contraction. Yeo et al. reported that YC-1 administration in xenograft immunodeficient mice blocked angiogenesis and inhibited tumor growth [ 35 ]. ARRY-797 ( Fig. 6 ) is a highly potent (IC 50 = 4.5 nM) and selective p38α (MAPK14) kinase inhibitor. The molecule was developed by Array BioPharma (a Pfizer subsidiary since mid-2019) for its therapeutic application against inflammation and pain. Currently, clinical trials are being conducted with this molecule to evaluate its therapeutic efficacy in humans. Array BioPharma has recently recruited patients to conduct a phase 2 clinical trial against Laminrelated dilated cardiomyopathy ( NCT02351856 ) [ 36 ]. Lamin A-related-dilated cardiomyopathy is connected with arrhythmias, myocardial remodeling, and dilatation of the left ventricle (LV), which leads to poor cardiac function and heart failure. In another study, i.e., phase 3 clinical trial, the company is recruiting patients with symptomatic dilated cardiomyopathy due to Lamin A/C (LMNA) gene mutation ( NCT03439514 ) [ 37 ]. Muchir et al. reported that ARRY-371797 treatment prevented left ventricular dilatation in Lmna H222P/H222P mice [ 38 ]. Left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD) were significantly reduced, while fractional shortening was significantly improved after the treatment. ARRY-371797 significantly reduced the expression of myosin light chain 1a and actin alpha 2, which are involved in sarcomere organization. Furthermore, the study found that phosphorylated p38α (MAPK14) levels were reduced in hearts after the treatment when compared to the placebo group [ 38 ]. Indazole-Cl (In-Cl) (3-chloro-2-(4-hydroxyphenyl)-2H-indazol-5-ol), with a phenyl-2H-indazole core, is an estrogen receptor-β agonist ( Fig. 7 ) [ 39 ]. Indazole-Cl was found to be effective among a series of nonsteroidal compounds synthesized by the Katzenellenbogen group. In-Cl is effective in preventing atherosclerosis through activation of estrogen receptor (ER)-β. Atherosclerosis is characterized by the deposition of fatty material on the inner walls of arteries. It is one of the most common causes of coronary artery disease and carotid artery disease, responsible for heart attack and stroke, respectively [ 40 ]. Vascular smooth muscle cells (VSMCs), the major components of blood vessels, are affected by hypoxia. The hypoxic condition affects VSMC inflammation, proliferation, and migration, which contribute to vascular stenosis and the atherosclerotic process. Estrogen receptor (ER)-β plays a key role in preventing the inflammatory response in VSMCs [ 41 - 44 ]. Through the hypoxia-inducible factor (HIF)-1-dependent pathway, hypoxia increases the expression of macrophage migration inhibitory factor MIF in VSMCs [ 45 ]. Macrophage migration inhibitory factor is a well-known proinflammatory factor that plays a crucial role in the progression of atherosclerosis. Treatment with In-Cl significantly reduced the hypoxia-induced reactive oxygen species (ROS) production and migration of VSMCs. Besides In-Cl’s effect as an ERβ agonist, the molecule also reduces ROS production induced under hypoxic condition. Increased cell migration and invasion by hypoxia were dramatically decreased following treatment with In-Cl. In-Cl also suppresses the inflammation in a model of endometriosis [ 46 ]. In-Cl significantly reduced the hypoxia-induced increase in COX-2 mRNA expression and NF-kB activity, which is mainly responsible for the generation of proinflammatory cytokines [ 47 ]. Platelet-derived growth factor (PDGF) is a cation glycoprotein mainly derived from platelets and also present in damaged endothelial cells, transitional fibroblasts, macrophages, smooth muscle cells, and mesangial cells [ 48 ]. The PDGF family consists of four gene products, PDGF-A, PDGF-B, PDGF-C, and PDGF-D, which selectively signal through two PDGF receptors (α,ß) to regulate diverse cellular functions. PDGF is a dimeric molecule consisting of disulfide-bonded A, B, C, and D polypeptide chains, and exists in homodimeric (PDGF-AA, BB, CC, and DD) as well as heterodimeric (PDGF-AB) isoforms [ 49 - 53 ] Gallini et al. reported that PDGFA overexpression led to severe fibrosis, resulting in lethal cardiac failure. In contrast, PDGFB overexpression resulted in focal fibrosis and moderate cardiac hypertrophy [ 54 ]. When treated with estrogen receptor agonists, proliferation-related genes that are upregulated in hypoxia are down-regulated. In the hypoxic state, the PDGF B gene is elevated by 1.9-fold. However, after treatment with In-Cl, it is reduced by 0.78-fold. The indazole molecule, In-Cl, has shown axon myelination and neuroprotection in experimental autoimmune encephalomyelitis (EAE) in vivo studies [ 55 ]. Pang et al. reported that in patients with acute coronary syndrome, there are considerably higher levels of PDGF in their peripheral and coronary artery blood [ 48 ]. The serum PDGF concentration in individuals with the acute coronary syndrome was significantly increased, notably in the coronary artery, and it was linked to the severity of coronary artery disease ( Fig. 8 ). Heldin et al. reviewed that in mouse models, PDGF-AA or PDGF-BB in the heart has been demonstrated to cause cardiac fibrosis or elevated collagen deposition by interstitial cardiac fibroblasts. Pang et al. have shown that PDGF-BB contributes to the development of atherosclerosis through chemotaxis. Therefore, targeting PDGF with indazole derivatives could be the future strategy to reduce cardiovascular diseases. 7-Nitroindazole is a heterocyclic indazole ring having a nitro group at position C7 ( Fig. 9 ). The molecule inhibits neuronal nitric oxide synthase in a selective manner. 7-nitroindazole had an anti-hypertrophic effect on the heart and decreased wall thickness of thoracic aorta and carotid arteries. However, the long-term 7-nitroindazole treatment caused adverse effects like pressure-independent cardiac hypotrophy as well as arterial wall hypotrophy and reduced contractile efficiency due to decreased endothelial and smooth muscle cell mass [ 56 , 57 ]. Administration of 7-nitroindazole markedly decreased the asymmetric dimethylarginine (ADMA) levels in the plasma of young spontaneously hypertensive rats (SHR). ADMA has been recognised to cause several adverse effects on cardiovascular disease. Chronic administration of 7-nitroindazole decreased homocysteine levels from 7.37 ± 0.467 μmol/l of control value to 4.890 ± 1.186 μmol/l. Plasma aspartate transaminase activities were reduced in the 7-nitroindazole-treated SHR group when compared to untreated controls. Administration of 7-nitroindazole improved the plasma redox status in comparison to both control and the NG-nitro-L-arginine methyl ester (NOS inhibitor) treated groups of animals. 7-nitroindazole treatment actively influenced cardiovascular biomarkers related to oxidative stress. 7-nitroindazole administration decreases ROS, homocysteine, and dimethylarginine levels. Similar to 7-nitroindazole, administration of another NOS inhibitor to young SHR led to a decrease in ADMA levels by 70%. It is known that higher production of homocysteine may induce oxidative stress, leading to elevated ADMA concentrations [ 58 ]. 7-nitroindazole treatment improves ROS, total thiol level (TTL), and the redox status i.e. ratio TTL/ROS [ 59 ]. 7-nitroindazole selective nNOS inhibition had no effect on total LV myocardial blood flow or its distribution in the subendocardial and subepicardial tissue layers [ 60 ]. Long-term 7-nitroindazole treatment resulted in pressure-independent cardiac hypotrophy as well as arterial wall hypotrophy and reduced contractile efficiency due to decreased endothelial and smooth muscle cell mass [ 56 , 57 ]. Administration of 7-nitroindazole to rats has shown dose-dependent inhibition of penile erection. The high-dose group had a significantly decreased maximal rise in intracavernous pressure (mICP) when compared to the low-dose group [ 61 ]. Boblewski et al. developed a novel series of centrally acting agents and evaluated their effect on blood pressure and heart rate in rats. They synthesized two novel marsanidine analogues that decrease blood pressure and heart rate in rats. These two molecules are 7-chloro-1-[(4,5-dihydro-1H-imidazol-2-yl)methyl]-1H-indazole (TCS-80) and 1-[(imidazolidin-2-yl)imino]-1H-indazole (marsanidine) ( Figs. 10 and 11 ). Among all of these analogues, compound TCS-80 had the greatest affinity for the I 1 -imidazoline receptors [ 62 ]. The adrenergic system controls neuronal, cardiovascular, endocrine, and metabolic functions [ 63 ]. α 2 -adrenoceptors (α 2 -ARs) are G-protein coupled receptors that mediate many of the central and peripheral actions. α 2 -ARs are present as three subtypes, α 2 A, α 2 B, and α 2 C ARs [ 64 ]. α 2 -ARs are involved in a variety of pathophysiological processes and regulate sympathetic nervous system activity, arterial blood pressure, body temperature, insulin secretion, and gastrointestinal motility [ 65 ]. Wasilewska et al. modified the selective α 2 -ARs agonists, i.e., 1-[(imidazolidin-2-yl)imino]-1 H- indazole (marsanidine, Fig. 11A ) and its methylene analogue 1-[(4,5-dihydro-1 H -imidazol-2-yl)methyl]-1 H -indazole ( Fig. 11B ) by introducing a fluorine group at different positions on the indazole ring. It is reported that the compound having fluorine at C7 ( Fig. 11C ) showed the highest hypotensive and bradycardic activities in in vivo cardiac model [ 66 ]. Structure-activity relationship (SAR) analysis showed that substituting a chlorine or methyl group at the C7 atom of the indazole nucleus of marsanidine resulted in compounds with higher cardiovascular activity than the congeners [ 66 - 69 ].